This project has two primary aims. The first aim is to understand how proteins that are destined to travel through the secretory pathway are targeted to transport sites in the endoplasmic reticulum (ER) or the bacterial inner membrane (IM). For the last decade we have been investigating the role of a ribonucleoprotein called the signal recognition particle (SRP) and its membrane-bound receptor in this process. Although SRP was initially believed to exist only in eukaryotic cells, the sequencing of a large number of microbial genomes has demonstrated that the particle is found in most (if not all) organisms. Previous studies have shown that in mammalian cells SRP recognizes the "signal sequences" found on virtually all secreted and membrane proteins as they emerge during translation and then catalyzes their translocation across the ER membrane upon interaction with the SRP receptor. A few years ago we demonstrated that bacterial SRP has a somewhat more restricted function in that it only targets integral membrane proteins to the IM. Consistent with the work of other laboratories, we found that most secreted proteins, by contrast, are targeted to the IM by molecular chaperones. In recent studies we have continued to analyze the mechanism by which SRP recognizes the highly diverse family of signal sequences found on different proteins and then releases them at the surface of the ER (or IM) in a regulated fashion. In the past year we have focused on the role of SRP RNA in signal sequence recognition. Although previous work has shown that signal peptides bind to SRP primarily via hydrophobic interactions with the 54 kD protein subunit, a recent crystallographic study raised the surprising possibility that electrostatic interactions between basic amino acids in signal peptides and the phosphate backbone of SRP RNA may also play a role in signal sequence recognition. To test this possibility, we examined the degree to which basic amino acids in a signal peptide influence the targeting of two E. coli proteins, maltose binding protein and OmpA. Whereas both proteins are normally targeted to the inner membrane by the molecular chaperone SecB, we found that replacement of their native signal peptides with another moderately hydrophobic but unusually basic signal peptide ("DEspP") rerouted them into the SRP pathway. Reduction in either the net positive charge or the hydrophobicity of the DEspP signal peptide decreased the effectiveness of SRP recognition. A high degree of hydrophobicity, however, compensated for the loss of basic residues and restored SRP binding. Taken together, the data suggest that the formation of salt bridges between SRP RNA and basic amino acids facilitates the binding of a distinct subset of signal peptides whose hydrophobicity falls slightly below a threshold level. The second aim of the project is to elucidate the function of factors that facilitate the transport of proteins across or insertion into the ER or bacterial IM. In the last year we have studied the structure of YidC, a highly conserved bacterial protein that has been shown to play an important but still poorly defined role in membrane protein biogenesis. Despite a striking sequence homology to a mitochondrial protein called Oxa1p, YidC has been reported to exist primarily as a monomer whereas Oxa1 has been reported to form a larger homooligomer. In an effort to explain this apparent disparity, we reexamined the quaternary structure of E. coli YidC. Using several different biochemical methods, we have obtained considerable evidence that YidC is a tetramer or larger oligomer. These results suggest that YidC is a multisubunit complex like most other protein conducting channels.